Purification and Properties of Phospholipase D from Actinomadura sp. Strain No. 362

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An extracellular phospholipase D from Actinomadura sp. Strain No. 362 was purified about 430-fold from the culture filtrate. The purified enzyme preparation was judged to be homogeneous on polyacrylamide gel electrophoresis. The molecular weight and isoelectric point of the enzyme were estimated to be about 50, 000-60, 000 and 6.4, respectively. The enzyme was most active at pH 5.5 and 50°C in the presence of Triton X-100, but showed the highest activity at pH 7.0 and 60-70°C in its absence. The enzyme was stable up to 30°C at pH 7.2 and also stable in the pH range of 4.0 to 8.0 on 2hr incubation at 25°C. With regard to substrate specificity, this enzyme hydrolysed lecithin best among the phospholipids tested. It was activated by Fe³⁺, A1³⁺, Mn²⁺, Ca²⁺, diethyl ether, sodium deoxycholate and Triton X-100, but was inhibited by cetyl pyridinium chloride and dodecylsulfate.